by Neal Chan
mentor: Benjamin Wolfe, Biology; funding source: Nelson Family Summer Scholars FundNCHAN_summer_scholars_poster
This summer I had the privilege of studying the effect of bacteria on flatworm regeneration. Because of the remote setup, my research focused on analyzing the genetic data of both the flatworm Dugesia japonica, and the bacteria that live on it.
Researchers use flatworms, like D. japonica and many other species, as models for tissue regeneration. Planarians can completely regenerate the entire body plan from just small fragments amputated from an intact worm. After an injury, cells will proliferate and migrate to the sight to form a blastema. From there, specialized stem cells differentiate, communicate, and divide rapidly to form complex structures and eventually an entire head and tail. Researchers are still figuring out how the flatworm possesses the biological memory to know what to grow after amputation.
Bacterial communities play an essential role in human biology, providing nutrients, influencing behavior, breaking down nutrients, resisting pathogens, reducing gut and skin tissue permeability, and many more. I am interested in the role of bacteria in D. japonica regeneration, both as inhibitors and promoters. To date we have recorded two species of bacteria to inhibit D. japonica regeneration, though we have isolated ten species from worms and found a consistent microbial community between colonies of worms.
This summer we wanted to learn more about the genomes of the bacteria—what systems do they possess to allow them to live and colonize worms? Since we had several species of bacteria, we wanted to know how these systems encoded by the microbes’ genes vary across species
On the host side, we wanted to know how the flatworm responded to the presence of bacteria while regenerating. What genes are expressed more or less when microbes are added?
A thorough genome search of the ten species of bacteria screened for genes that might be involved in colonization of worm tissue. This includes flagella and pili for motility and immunological interactions, mucin-degrading enzymes to break down the thick mucin layers that D. japonica possess to resist pathogens, Type III/VI secretion systems, and collagenases. We found many annotated genes related to these structures as well as a great deal of genetic variation between the species of bacteria. The bacterial community is likely composed of some motile—capable of motion—species, and contrasted with species with more mucin-degrading enzymes. From this data it’s possible that species fit niches in the community, playing roles in processes we have not yet considered.
Logically, our next step this summer was to look into how the flatworm responded to the presence of bacteria. We already knew that two bacteria, Aquitalea sp. F5 and Pseudomonas sp. KBW10 inhibit regeneration. Previous to this summer, these two bacteria were applied to regenerating worms. The mRNA from these worms were extracted and analyzed; the RNA can tell us which genes are being turned on—or upregulated—and which are being turned off—downregulated—in response to bacterial colonization.
We found that D. japonica gene expression undergoes profound changes in the presence of Aquitalea sp. F5, with upwards of 600 differentially expressed genes (DEGs). The other inhibitory bacterium, Pseudomonas, also affects gene regulation. Another striking finding was the identification of 48 genes that were differentially expressed in both the Aquitalea and Pseudomonas species. Another three genes were differentially expressed in the Aquitalea, Pseudomonas, and the control bacterium treatment. I looked into the previous studies of these three genes, and found that they are all involved in the immune response to pathogens. This seems to indicate that there is a conserved immune response by D. japonica in response to bacterial colonization. This semester I’m working to validate the importance of these genes by RT-qPCR.